The present invention relates to improvements in apparatus for differentiating small particles of various types on the basis of their respective light scattering characteristics. The invention is particularly useful in hematology instruments for differentiating various types or classes of blood cells.
The use of light scattering measurements as a means for differentiating various types of small particles is well known. For example, in virtually all sophisticated hematology instruments, it is common to measure the forward light scattering properties of blood cells by passing the cells, one at a time, through the interrogation zone of an optical flow cell. While in the interrogation zone, each cell is irradiated by a laser beam, and one or more photodetectors, strategically positioned forward of the interrogation zone, operate to sense the level of forward scattered radiation, often within different predetermined angular ranges. In addition to measuring forward light scatter, some hematology instruments measure side scatter as well, using a separate photodetector located laterally of the irradiated cell. These light scattering measurements are often combined with other simultaneously made measurements, e.g., axial light-loss, DC volume and/or RF conductivity measurements, to better differentiate cell types of particular interest from other cells and particulate material in the sample that have similar light-scattering properties within the measurement ranges. Having made the various parameter measurements, the instrument then produces scattergrams in which the different parameters measured are plotted against each other. Ideally, each cell type appears on these scattergrams as a tight cluster of data points, each point representing an individual cell, and each cluster being readily identifiable by a clearly identified spacing from other clusters of data points. In such case, it is a relatively simple matter to xe2x80x9cgatexe2x80x9d cells of one cluster from those of another cluster and to enumerate the cells of each type. This ideal, unfortunately, is sometimes difficult to realize since, for a variety of reasons, a certain percentage of cells of one type invariably invade the spatial domain of cells of other types, making the differentiation of each type somewhat imprecise.
To more precisely differentiate blood cells and the like on the basis of their light-scattering signature, various photodetector configurations have been proposed. As noted above, it is often desirable to measure light scatter within different angular ranges. To effect such measurements, some photodetectors comprise a series of concentric rings or, more commonly, ring segments of light-sensitive material, typically PIN diode material. The rings or segments thereof are positioned in a plane forward of the cell interrogation zone with the ring center coinciding with the axis of the cell-irradiating beam. The spacing between the detector plane and the interrogation zone, together with the radial position and width of each ring determines the angular range within which forward light scatter is measurable. Such a photodetector configuration is disclosed, for example, in U.S. Pat. No. 6,232,125 to Deka et al. In such a detector configuration, the area of the light-sensitive material of each detector ring or arc increases with ring diameter. So, too, does the sensitivity of the ring due to the increased area of the photodetector material. This increased sensitivity is desirable from the standpoint that the light scatter intensity (on average) decreases dramatically with increasing scatter angle. But, the increasing detector size from ring to ring with increasing angle results in an undesirable decrease in detector response time, the latter being inversely proportional to the detector""s active area.
It has been suggested that multiple bundles of fiber optics, arranged in concentric rings, can be used to optically couple scattered radiation from a scatter plane to multiple photodetectors (e.g., photomultiplier tubes and photodiodes) remotely spaced from the scatter plane. See, xe2x80x9cCell Differentiation Based on Absorption and Scatteringxe2x80x9d by Wolfgang G. Eisert, The Journal of Histology and Cytochemistry, Vol.27, No.1, pp404-409 (1979). As described by Eisert, optical fibers are arranged so that their respective light-collecting ends form five concentric rings centered about a centrally located light-collecting bundle of optical fibers. The respective distal ends of the individual fibers of each of the five concentric rings are optically coupled to five different photomultiplier tubes, and the distal ends of the individual fibers of the center bundle are optically coupled to a photodiode. Thus, each ring of fibers collects scattered light in a discrete angular range determined by the diameter of the fiber (or the width of the rings), the radial displacement of the fiber end relative to the beam axis, and the axial spacing of the fiber ends from the scattering light source. The center bundle of fibers is optically aligned with the beam axis, and the other bundles, with their individual fibers being arranged in a circle, are also arranged parallel to the beam axis. The center bundle of fiber optics, being positioned on the beam axis, serves to monitor the axial light loss of the beam, as occasioned by the passage of cells therethrough.
In the fiber-optic light coupler proposed by Eisert above, the respective light-collecting ends of all the fibers are disposed in a common plane that is arranged perpendicular to the optical axis of the cell-irradiating light beam. Thus, it will be appreciated that, due to the numerical aperture of the fibers, the optical coupling of scattered light into the optical fibers deteriorates as the scatter angle increases. Additionally, as the scatter angle increases, the angle of incidence between the scattered light and the fiber end increases, thereby increasing the number of internal reflections required to transmit the scattered light from one end of the fiber to the other end. This problem of coupling efficiency is exacerbated by the dramatic reduction in scatter intensity at relatively large scatter angles. Further, the presence of the respective fiber ends of the central bundle of fibers in the scatter-detection plane can be problematic from a retro-reflection standpoint, i.e., the fiber ends tend to reflect a significant percentage of the relatively intense cell-irradiating light beam backwards, towards the optical flow cell used to control the movement of cells. Upon being reflected again by the flow cell surface, the re-reflected light will be collected by the fiber ends surrounding the central bundle, the result being that the relatively low level light scatter signals from the cells of interest are swamped out.
In view of the foregoing discussion, an object of this invention is to provide an apparatus for more efficiently coupling light scattered from, or otherwise modulated by, an irradiated particle (e.g., a blood cell) to a photodetector in an apparatus adapted to differentiate such particles on the basis of their respective light-scattering signatures.
In accordance with a preferred embodiment of the invention, apparatus for detecting light scatter from an irradiated particle of interest comprises (a) an optically transparent flow cell having a cell-interrogation zone through which particles of interest can be made to pass, one at a time, while a beam of optical radiation irradiates each particle; and (b) a plurality of optical fibers that operate to receive scattered light from the irradiated particles and to transmit such light, via multiple internal reflections, to one or more remote photodetectors for detection. According to the invention, a portion of each fiber in the vicinity of its light-collecting end is supported so that the optical axis of the supported fiber portion extends towards the scattering source (i.e., the irradiated particle), and most preferably towards the apparent position of the scattering source, taking into account the refractive effects of the optical flow cell wall through which the light scatter is collected. As a result of this arrangement, the scattered light is efficiently coupled into the fibers and transmitted through the fibers with minimal losses. Preferably, one end of each optical fiber (i.e., its light-collecting end) is disposed on a spherically-concave surface of an optical fiber holder that serves to optimally position the fibers for light-collection. In use, the concave surface is arranged so that its center of curvature is located proximate the apparent position of the light-scattering particles, again as viewed through an optically transparent wall of the optical flow cell. Preferably, the light-collecting optical fiber ends are arranged on the concave surface to form one or more circular or arcuate patterns with the center of the circle(s) or arcs coinciding with the axis of the particle-irradiating beam. Preferably, additional optical fibers are supported by the fiber optic holder in positions to collect axial beam light as modulated in intensity by particles passing through the beam path, and as diffused by a light-diffusing surface within the fiber optic holder that minimizes retro-reflection of the beam light into regions that may degrade the signal-to-noise level of the detected light signals.
The invention and its advantages will be better understood from the ensuing detailed description of preferred embodiments, reference being made to the accompanying drawings in which like reference characters denote like parts.